In vitro experimental study on the killing effect of EGFRvⅢ-targeted CAR-NK cells on glioma cells
Objective To explore the specific killing effect of human peripheral blood-derived NK cells modified with Chimeric Antigen Receptor(CAR)targeting epidermal growth factor receptor type Ⅲmutant(EGFRv Ⅲ)on glioma cells.Methods Human peripheral blood-derived NK cells were obtained by Ficoll density gradient centrifugation and cultured in vitro.CAR-NK cells targeting EGFRvⅢand U87-EGFRvⅢ stable cell lines were constructed by lentivirus infection.The experiments were divided into NK+U87 group,NK+U87-EGFRvⅢ group,CAR-NK+U87 group and CAR-NK+U87-EGFRvⅢgroup.The killing effect of CAR-NK cells on glioma cells was evaluated by real-time unlabeled cell analysis(RTCA)and lactate dehydrogenase(LDH)release assay.At the same time,enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),granzyme B(GzmB)and perforin 1(PRF1)in cell culture supernatant.Results Flow cytometry analysis showed that the purity of human peripheral blood-derived NK cells was 97.6％,and the survival rate was 98.2％.The second generation of CAR molecules targeting EGFRv Ⅲwas successfully constructed,and CAR-NK cells targeting EGFRv Ⅲ were successfully obtained.Flow cytometry analysis showed that the positive cell rate was 69.6％.RTCA results showed that the growth curve of target cells increased with time,and the proliferation rate of U87 cells overexpressing EGFRv Ⅲwas higher than that of U87 cells.After adding effector cells,the cell index showed a downward trend.At the same effector-target ratio(E∶T),the cell index of CAR-NK+U87-EGFRv Ⅲ group decreased faster than that of NK+U87-EGFRvⅢ group,and faster than that of CAR-NK+U87 group.LDH release assay further demonstrated that the cleavage rate of target cells gradually increased with the increase of effector-target ratio(FNK+U87=700.61,FCAR-NK+U87=2 063.97,FNK+U87-EGFRvⅢ=5707.00,FCAR-NK+U87-EGFRvⅢ=28 858.57,all P＜0.05).At E∶T=2∶1 and E∶T=3∶1,the cell lysis rates of NK+U87-EGFRvⅢgroup and CAR-NK+U87 group were lower than that of CAR-NK+U87-EGFRv Ⅲ group(all P＜0.05).In addition,ELISA results showed that the secretion of TNF-α,IFN-γ,GzmB and PFR1 increased with the increase of effect-target ratio in the same group(all P＜0.05);under the same effect-target ratio,except at E:T=1:1,there was no significant difference in GzmB secretion between CAR-NK+U87-EGFRv Ⅲ group and CAR-NK+U87 group(P＞0.05);the secretion of TNF-α,IFN-γ,GzmB and PFR1 in CAR-NK+U87-EGFRv Ⅲ group increased compared with NK+U87-EGFRv Ⅲ group and CAR-NK+U87 group respectively(all P＜0.05).Conclusion CAR-NK cells targeting EGFRvⅢ have a strong killing effect on glioma cells with positive expression of EGFRv Ⅲ,and their anti-tumor ability is better than that of NK cells.
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